Proteins which bind without base or sequence specificity to single-stranded DNA (ssDNA) and have no demonstrable enzymatic activity have now been isolated from a large number of prokaryotic and eukaryotic organisms. While several prokaryotic DNA binding proteins have been well characterized, there is no structural data available on any eukaryotic single-stranded DNA binding protein and the in vivo functions of this class of proteins are not well understood. We propose to obtain preliminary structural data on the ssDNA binding protein from rat liver and use this information to audit the results of DNA sequencing studies (being done in collaboration with this project) on the gene encoding this protein. Functional domains will be located within the resulting primary structure by characterizing two subtilisin cleavage products derived from the rat liver ssDNA binding protein. Comparative tryptic peptide mapping by HPLC will be used to determine the extent of homology between the ssDNA binding proteins isolated from normal and regenerating rat liver on the one hand and an immunologically cross-reacting protein from Drosophila on the other. In addition to our studies on the rat liver protein we propose to develop procedures to isolate each of the 5 subspecies that are present in highly purified ssDNA binding protein preparations from calf thymus. The molecular basis for this microheterogeneity will be determined and the low molecular weight (26,000 dalton) species will be sequenced by solid phase Edman degradation. Limited proteolysis will be used to localize functional domains and a radioimmune assay will be developed to facilitate identification of homologous proteins in other species and to determine the tissue distribution of this ssDNA binding protein. The proposed research is basic to our understanding of those processes (DNA replication, transcription, translation) which require a single-stranded nucleic acid template.